Pesticidal toxins and nucleotide sequences which encode...

Details Pesticidal toxins and nucleotide sequences which encode... Pesticidal toxins and nucleotide sequences which encode...

1998-05-06

2001-06-05

Bugalsky, Gabriele E. (Department: 1653)

Multicellular living organisms and unmodified parts thereof and

Plant, seedling, plant seed, or plant part, per se

C800S298000, C800S302000, C435S252300, C435S410000, C435S418000, C536S023710

Reexamination Certificate

active

06242669

ABSTRACT:

BACKGROUND OF THE INVENTION
The soil microbe
Bacillus thuringiensis
(B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopicallyas distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering these B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, F. H., L. Kim [1988]
TIBTECH
6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.
Until the last fifteen years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of
B. thuringiensis
subsp.
kurstaki
have been used for many years as commercial insecticides for lepidopteran pests. For example,
B. thuringiensis
var.
kurstaki
HD-1 produces a crystalline &dgr;-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely
israelensis
and
morrisoni
(a.k.a.
tenebrionis
, a.k.a. B.t. M-7, a.k.a. B.t.
san diego
), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] “Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms,” in
Controlled Delivery of Crop Protection Agents
, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255.). See also Couch, T. L. (1980) “Mosquito Pathogenicity of
Bacillus thuringiensis
var.
israelensis,” Developments in Industrial Microbiology
22:61-76; and Beegle, C. C. (1978) “Use of Entomogenous Bacteria in Agroecosystems,”
Developments in Industrial Microbiology
20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter (1983)
Z. ang Ent.
96:500-508 describe
Bacillus thuringiensis
var.
tenebrionis
, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle,
Leptinotarsa decemlineata
, and
Agelastica alni.
More recently, new subspecies of B.t. have been identified, and genes responsible for active &dgr;-endotoxin proteins have been isolated (Höfte, H., H. R. Whiteley [1989
] Microbiological Reviews
52(2):242-255). Höfte and Whiteley classified B.t. crystal protein genes into four major classes. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne, L. Kim [1992]
Bio/Technology
10:271-275). CryV has been proposed to designate a class of toxin genes that are nematode-specific. Lambert et al. (Lambert, B., L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J. Seurinck, K. van Audenhove, J. Van Rie, A. Van Vliet, M. Peferoen [1996]
Appl. Environ. Microbiol
62(1):80-86) describe the characterization of a Cry9 toxin active against lepidopterans. Published PCT applications WO 94/05771 and WO 94/24264 also describe B.t. isolates active against lepidopteran pests. Gleave et al. ([1991] JGM 138:55-62), Shevelev et al. ([1993]
FEBS Lett.
336:79-82; and Smulevitch et al. ([1991]
FEBS Lett.
293:25-26) also describe B.t. toxins. Many other classes of B.t. genes have now been identified.
The cloning and expression of a B.t. crystal protein gene in
Escherichia coli
has been described in the published literature (Schnepf, H. E., H. R. Whiteley [1981
] Proc. Natl. Acad. Sci. USA
78:2893-2897.). U.S. Pat. Nos. 4,448,885 and 4,467,036 both disclose the expression of B.t. crystal protein in
E. coli
. U.S. Pat. Nos. 4,990,332; 5,039,523; 5,126,133; 5,164,180; and 5,169,629 are among those which disclose B.t. toxins having activity against lepidopterans. PCT application WO96/05314 discloses PS86W1, PS86V1, and other B.t. isolates active against lepidopteran pests. The PCT patent applications published as WO94/24264 and WO94/05771 describe B.t. isolates and toxins active against lepidopteran pests. B.t. proteins with activity against members of the family Noctuidae are described by Lambert et al., supra. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose
B. thuringiensis
strain
tenebrionis
which can be used to control coleopteran pests in various environments. U.S. Pat. No. 4,918,006 discloses B.t. toxins having activity against dipterans. U.S. Pat. Nos. 5,151,363 and 4,948,734 disclose certain isolates of B.t. which have activity against nematodes. Other U.S. patents which disclose activity against nematodes include U.S. Pat. Nos. 5,093,120; 5,236,843; 5,262,399; 5,270,448; 5,281,530; 5,322,932; 5,350,577; 5,426,049; 5,439,881, 5,667,993; and 5,670,365. As a result of extensive research and investment of resources, other patents have issued for new B.t. isolates and new uses of B.t. isolates. See Feitelson et al., supra, for a review. However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art.
Isolating responsible toxin genes has been a slow empirical process. Carozzi et al. (Carozzi, N. B., V. C. Kramer, G. W. Warren, S. Evola, G. Koziel (1991)
Appl. Env. Microbiol.
57(11):3057-3061) describe methods for identifyingtoxin genes. U.S. Pat. No. 5,204,237 describes specific and universal probes for the isolation of B.t. toxin genes. That patent, however, does not describe the probes and primers of the subject invention.
WO 94/21795, WO 96/10083, and Estruch, J. J. et al. (1996)
PNAS
93:5389-5394 describe toxins obtained from Bacillus microbes. These toxins are reported to be produced during vegetative cell growth and were thus termed vegetative insecticidal proteins (VIP). These toxins were reported to be distinct from crystal-forming &dgr;-endotoxins. Activity of these toxins against lepidopteran and coleopteran pests was reported. These applications make specific reference to toxins designated Vip1A(a), Vip1A(b), Vip2A(a), Vip2A(b), Vip3A(a), and Vip3A(b). The toxins and genes of the current invention are distinct from those disclosed in the '795 and '083 applications and the Estruch article.
BRIEF SUMMARY OF THE INVENTION
The subject invention concerns materials and methods useful in the control of non-mammalian pests and, particularly, plant pests. In one embodiment, the subject invention provides novel B.t. isolates having advantageous activity against non-mammalian pests. In a further embodiment, the subject invention provides new toxins useful for the control of non-mammalian pests. In a preferred embodiment, these pests are lepidopterans and/or coleopterans. The toxins of the subject invention include &dgr;-endotoxins as well as soluble toxins which can be obtained from the supernatant of Bacillus cultures.
The subject invention further provides nucleotide sequences which encode the toxins of the subject invention. The subject invention further provides nucleotide sequences and methods useful in the identification and characterization of genes which encode pesticidal toxins.
In one embodiment, the subject invention concerns unique nucleotide sequences which are useful as hybridization probes and/or primers in PCR techniques. The primers produce characteristic gene fragments which can be used in the identification, characterization, and/or isolation of specific toxin genes. The nucleotide sequences of the subject invention encode toxins which are distinct from

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